The purpose of this proposal is to define, in chemical terms, the basis for development of the capacity for motility as sperm traverse the bovine epididymis. The proposal has its origin in the development in our laboratory of an in vitro model system in which it has been possible to initiate progressive motility in immotile sperm from the bovine caput epididymis by simultaneous elevation of cAMP levels with high concentrations of phosphodiesterase inhibitors and addition of a specific epididymal protein, which we have called Forward Motility Protein (FMP). Recently, we have provided the first evidence that extracellular adenosine regulates cAMP levels and motility in mammalian sperm (most likely through an Ra plasma membrane receptor) and that the capacity to respond to adenosine increases markedly in bovine sperm during epididymal maturation. We have also provided the first evidence that a developmental change in the pH of the flagellar cytosol is required before maturational increases in cAMP are manifested as motility and that this pH change markedly increases the sensitivity of caput sperm to adenosine and dramatically lowers the levels of PDE inhibitors required to initiate motility. We have also recently described, by cinematography, the motility characteristics of caput sperm altered by impure preparations of FMP and have confirmed that these preparations change a number of motility parameters of caput sperm, including progressivity, to values approximating those of mature caudal sperm. In this proposal, we plan to document in detail the role of adenosine in the epididymal development of motility, provide chemical evidence for the existence of a membrane adenosine receptor, show that adenosine in luminal fluids does, in fact, regulate motility, measure the pH changes in sperm during development and after in vitro activation with bases, determine the mechanism(s) by which the pH change renders caput sperm "caudal-like", develop a computer-assisted assay with which to quantify the complex motility patterns of developing sperm in the presence and absence of FMP, use this assay to purify FMP, develop an RIA for FMP, use the RIA to study the epididymal binding site, and cellular binding site of the protein, and, finally determine whether protein carboxymethylation reactions are involved in motility initiation by virtue of their ability to affect cAMP metabolism.